Francisella tularensis is classified as an NIAID Category A agent because of its extreme infectivity, ease of dissemination, and substantial capacity to cause illness and death. Induction of both humoral and cellular immunity appears to be key for protection but there is currently no vaccine approved for human use and experts in the field have highlighted the need for new, localized immunostimulatory approaches. This Program Project brings together a diverse group of individuals with particular expertise in the fields of microbiology, cell biology, and mucosal immunology who will continue to explore in an integrated fashion, novel strategies for protection against pulmonary tularemia. Our group has made remarkable progress during the previous funding period, including the demonstration that vaccination with inactivated F. tularensis targeted to Fc receptors on lung antigen presenting cells or intranasal inoculation of an antioxidant mutant strain provides significant protection against the highly virulent SchuS4 strain. We will now build upon our achievements to: 1) Complete development of our promising and novel strategies for induction of anti-F. tularensis pulmonary immunity and determine the mechanisms that mediate protection. 2) Map the signaling networks responsible for coordinated intra- and extracellular recognition of F. tularensis. 3) Define the redox control of F. tularensis pathogenesis and its role in regulating immune protection. The overall goal of the Project is to obtain novel insights into pulmonary Francisella immunopathogenesis and to discover and refine new vaccination platforms that can be exploited to ultimately protect the human population against respiratory infection by the highly virulent type A strain, SchuS4. RELEVANCE: The overall goal of the Project continues to be development of vaccination platforms for effective immune protection at mucosal surfaces. The results of these studies will ultimately be used for novel mucosal vaccination against human respiratory infection with F. tularensis. Project 1 - Immune Protection against Pulmonary Tularemia Project Leader (PL): Metzger, Dennis W. (Description as provided by applicant) Our overall objective is to develop approaches for effective vaccination against pulmonary tularemia. During the previous funding period, we made considerable progress, including the demonstration that significant protection in C57BL/6 mice against the highly virulent type A strain of F. tularensis (Ft), SchuS4, can be induced by i.n. inoculation of FcR-targeted inactivated LVS (iFt). This is the first case to our knowledge in which an inactivated vaccine has provided protection against Ft SchuS4. In this renewal application, we will further optimize conditions for mucosal vaccination, characterize the effects of pulmonary vaccination, and define the cellular and humoral mechanisms responsible for protective immunity against the highly virulent type A strain, SchuS4. Specifically, we will: 1) Determine the ability of i.n. vaccination with FcR-targeted immunogens, in the absence or presence of exogenous IL-12, to enhance the immune response to, and levels of protection against, mucosal (i.n.) challenge with Ft SchuS4. Two separate approaches will be used to target iFt to FcR: a) administration of preformed mAb-iFt complexes, and b) simultaneous inoculation of uncomplexed iFt plus anti-Ft mAb; 2) Establish the mechanisms responsible for enhanced induction of immunity by assessing the distribution of FcR targeted iFt to tissues and lymphoid organs to determine if enhanced localization of iFt to secondary lymphoid tissues, and APC within these tissues, occurs as a result of FcR targeting. It will also be determined if enhanced iFt processing and presentation results from targeting to FcR on APC; and 3) Establish the effector mechanisms responsible for enhanced protection after i.n. vaccination with FcR-targeted bacteria. The mechanisms responsible for protection after mucosal vaccination will be investigated by passive transfer of anti-Ft antibody or cells to naive mice with particular attention paid to examining a potential requirement for synergy between humoral and cellular immune mechanisms for induction of effective protection. The roles of TLR/NLRs and ROS/RNS in both inductive and effector phases will be examined by using genetically deficient mice and specific agonists/antagonists, in consultation with the PLs of subprojects 2 and 3. The results of subproject 1 will allow the design of new mucosal vaccination strategies for effective biodefense against infection with virulent Ft and will provide novel insight into the pulmonary immune mechanisms that are responsible for protection against respiratory tularemia. RELEVANCE: The results of this subproject will allow the design of new mucosal vaccination strategies for effective biodefense against infection with virulent Ft and will provide novel insight into the pulmonary immune mechanisms that are responsible for protection against respiratory tularemia.